d.d. 16 augustus 2003: Hieronder verschillende studies, gepubliceerd in Nature, die bewijzen dat in bessen en rood fruit een grote hoeveelheid quercetin-C zit. Een vorm van vitamine-C die bewezen heeft in preventie maar ook in een behandeling van kanker een belangrijke rol te kunnen spelen. Maar misschien nog belangrijker en aanverwant lijkt ellagic acid, een stofje veel voorkomend in bessen en rode vruchten en noten. Als u naar Pubmed gaat en ellagic acid intypt krijgt u een grote hoeveelheid studies die bijna allemaal positieve effecten aangeven van ellagic- acid. Vooral voor kankersoorten als baarmoederhalskanker, prostaatkanker, eierstokkanker en borstkanker lijkt dit ellagic acid een goed effect te hebben. Op deze pagina kunt u 120 referenties van studies bekijken die allemaal lijken te bewijzen dat ellagic acid een uitstekend effect heeft op bacteriën, virale infecties enz.. De ellagic-website claimt dat ellagic-acid ook een effect heeft in een kankerbehandeling doordat het de groei van tumorcellen afremt, zo niet blokkeert waardoor automatisch een apoptoseproces - zelfmoord van kankercellen - ontstaat.

Ellagic-acid triggers many types of cancer cells (breast, cervical, colon, pancreatic, esophageal, skin, prostate, leukemia) into apoptosis; in grapes/raisins, red raspberries, strawberries, & walnuts; may require rather high dosages. Oftewel in een vrije Nederlandse vertaling: Ellagic-acid brengt verschillende soorten kankercellen ( borst-, baarmoederhals-, darm-, alvleesklier-, huid-, prostaat- en leukemiecellen) tot zelfmoord. Druiven/rozijnen, rode frambozen, aardbeien, noten bevatten grote hoeveelheden Ellagic-acid. Maar leest u maar eens de informatie hieronder , ik ben geen arts en sommige informatie is te wetenschappelijk voor me maar wellicht dat uw arts hier wel wat wijzer uit kan worden. Goed gekwalificeerde orthomoleculaire artsen schrijven , zie voor adressen onder orthomoleculaire artsen bijna altijd ellagic acid voor of in ieder geval Quercetin-C.

Bron: Nature

Abstract

Objective: To study serum quercetin concentrations of subjects consuming berries or habitual Finnish diets.

Design: Randomized parallel dietary intervention.

Subjects: Forty healthy men (age 60 y).

Intervention: Twenty subjects consumed 100 g/day of berries (black currants, lingonberries and bilberries) for 8 weeks. Twenty subjects consuming their habitual diets served as controls. Fasting blood samples were obtained 2 weeks prior to the study, at baseline, and at 2, 4 and 8 weeks. Intake of quercetin was assessed from 3 day food records collected at baseline and at 8 weeks.

Results: The serum quercetin concentrations were significantly higher in the subjects consuming berries compared to the control group (P=0.039 ANCOVA with repeated measures). During the berry consumption period the mean serum concentrations of quercetin ranged between 21.4 and 25.3 µg/l in the berry group, which was 32-51% higher compared with the control group. According to 3 day food records, there was no difference in quercetin intake at baseline, but at 8 weeks the intake was 12.3±1.4 mg/day (mean±s.e.m.) in the berry group and 5.8±0.6 mg/day in the control group (P=0.001).

Conclusions: The results indicate that the berries used in this study are a good source of bioavailable quercetin.

Sponsorship: The study was supported by the Academy of Finland, Juho Vainio Foundation and the Finnish Foundation for Cardiovascular Research.

European Journal of Clinical Nutrition (2003) 57, 37-42. doi:10.1038/sj.ejcn.1601513

Consumption of black currants, lingonberries and bilberries increases serum quercetin concentrations

I Erlund1,b, J Marniemi2,a,b, P Hakala2,b, G Alfthan1,b, E Meririnne3,b and A Aro1,a,b

1Biomarker Laboratory, National Public Health Institute, Helsinki, Finland

2Research and Development Centre, The Social Insurance Institution, Turku, Finland

3Research Unit of Substance Abuse, National Public Health Institute, Helsinki, Finland

Correspondence to: I Erlund, Biomarker Laboratory, National Public Health Institute (KTL), Mannerheimint. 166, 00300 Helsinki, Finland. E-mail: iris.erlund@ktl.fi


aGuarantors: AA and JM

bContributors: IE performed quercetin analyses, interpreted the data and wrote the article. PH and JM designed and executed the dietary intervention. PH calculated quercetin intake. EM performed statistical analyses. GA and AA supervised quercetin analyses. All authors read and commented on the manuscript.


Abstract


Objective: To study serum quercetin concentrations of subjects consuming berries or habitual Finnish diets.

Design: Randomized parallel dietary intervention.

Subjects: Forty healthy men (age 60 y).

Intervention: Twenty subjects consumed 100 g/day of berries (black currants, lingonberries and bilberries) for 8 weeks. Twenty subjects consuming their habitual diets served as controls. Fasting blood samples were obtained 2 weeks prior to the study, at baseline, and at 2, 4 and 8 weeks. Intake of quercetin was assessed from 3 day food records collected at baseline and at 8 weeks.

Results: The serum quercetin concentrations were significantly higher in the subjects consuming berries compared to the control group (P=0.039 ANCOVA with repeated measures). During the berry consumption period the mean serum concentrations of quercetin ranged between 21.4 and 25.3 µg/l in the berry group, which was 32-51% higher compared with the control group. According to 3 day food records, there was no difference in quercetin intake at baseline, but at 8 weeks the intake was 12.3±1.4 mg/day (mean±s.e.m.) in the berry group and 5.8±0.6 mg/day in the control group (P=0.001).

Conclusions: The results indicate that the berries used in this study are a good source of bioavailable quercetin.

Sponsorship: The study was supported by the Academy of Finland, Juho Vainio Foundation and the Finnish Foundation for Cardiovascular Research.

European Journal of Clinical Nutrition (2003) 57, 37-42. doi:10.1038/sj.ejcn.1601513


Keywords

human; dietary intervention; berries; quercetin; flavonoids

Introduction

Flavonoids are polyphenolic compounds widely occuring in plants. One of the most studied flavonoids is the flavonol quercetin. The compound exhibits a wide range of biological activities, such as antioxidative (Aviram & Fuhrman, 1998; Chopra et al, 2000), anticarcinogenic (Pereira et al, 1996; Caltagirone et al, 1997) and enzyme-inhibiting (Siess et al, 1995; Peet & Li, 1999) activities. Furthermore, although the results are somewhat controversial, several epidemiological studies indicate a protective effect for quercetin on cardiovascular disease (Hertog et al, 1995; Knekt et al, 1996; Yochum et al, 1999).

According to data from the Seven Countries Study, the main dietary sources of quercetin are onions, tea, apples and red wine (Hertog et al, 1995). However, in Nordic countries, such as Finland, where berries are commonly consumed, berries are a more important source of quercetin than, for instance, red wine (Hirvonen, 2001). In Finland, the berries contributing most significantly to the total intake of quercetin are lingonberries (Vaccinium vitis-idaea), which are closely related to cranberries (Vaccinium oxycoccus), bilberries (Vaccinium myrtillus), which are closely related to blueberries, and black currants (Ribes nigrum; Häkkinen et al, 1999). Quercetin concentrations of 74-146 mg/kg have been found in lingonberries (Häkkinen et al, 1999; Mattila et al, 2000), in black currants the concentration ranges between 52 and 122 mg/kg (Mikkonen et al, 2001), and a concentration of 30 mg/kg has been reported in bilberries (Häkkinen et al, 1999).

Quercetin is mainly present in plants as glycosides and different plants contain different quercetin glycosides. Onions, for instance, contain quercetin glucosides, while in lingonberries the compound is present at least as arabinosides and rutinosides. No data is available on the bioavailability of quercetin from berries or some of the quercetin glycosides present in berries. Previous supplementation studies have shown that quercetin is bioavailable from foods such as onions and apples (Hollman et al, 1997), tea and red wine (de Vries et al, 2001), and from capsules containing quercetin aglycone or quercetin-3-rutinoside (Erlund et al, 2000). The bioavailability of the compound and the site of absorption in the gastrointestinal tract seems to depend on the type of sugar it is bound to. Quercetin from quercetin glucosides from onions are rapidly and efficiently absorbed from the proximal parts of the small intestine (Hollman et al, 1997), while quercetin from quercetin-3-rutinoside is absorbed from the distal parts of the small intestine or the colon (Erlund et al, 2000). In a pharmacokinetic study the bioavailability of quercetin from quercetin-3-rutinoside varied remarkably between individuals and was poorest in men (Erlund et al, 2000, 2001).

The aims of the present study were to determine the impact of daily consumption of 100 g of berries (black currants, lingonberries and bilberries) on serum quercetin concentrations in healthy middle-aged men, and to study serum quercetin concentrations in subjects consuming their habitual diets. Indices of antioxidant capacity were also measured from the samples, and the results have been published previously (Marniemi et al, 2000).


Subjects and methods

Subjects

The study population consisted of 60 male volunteers, all 60 y of age, living in the city of Turku. The subjects were checked to be in good health and were free of medication. The subjects were asked to restrain from dietary supplements for one month prior to and during the study. Their weights were within the normal range or their overweight was less than 20% (body mass index (BMI)<30 kg/m2).

The subjects were randomized into three groups (n=20 in each group; Marniemi 2000). One group received berries, one group vitamin supplements, and one group served as a control group. Subjects from the berry and the control groups were included in this study (n=40). The vitamin supplements did not contain quercetin, and therefore serum samples from that group were not analyzed for quercetin.

Study design

The subjects in the berry group were given 2 kg each of deep-frozen black currants, lingonberries and bilberries. The berries were packed in 100 g portions in plastic bags. The subjects were instructed to take one bag out of the freezer each day and eat one portion of berries per day. They were also instructed to eat the different berries in turns to ensure an even distribution over the 8 week intervention period. The berries were eaten fresh and heating of the berries was not allowed. The control group received 500 mg daily of calcium gluconate. It is unlikely that calcium affects the absorption of quercetin (Hollman et al, 2001). The subjects were instructed not to alter their normal dietary habits during the study. The study protocol was approved by the Ethics Committee of the Social Insurance Institution and all subjects gave their informed written consent prior to participation.

The subjects were asked to record any deviations from instructions regarding diet and berry consumption. Compliance was emphasized and each subject was asked about it separately when they came in for the blood samplings.

Intake of quercetin

The subjects filled out 3 day dietary records right before the beginning of the study and at 8 weeks. The average daily intakes of quercetin were calculated with the Nutrica computer program developed at the Social Insurance Institution (Knuts et al, 1987). The database of this program has been validated by Hakala et al (1996). Quercetin data from the Fineli database (provided by M-L Ovaskainen from the National Public Health Institute) were added to the Nutrica database.

Blood sampling and chemical analyses

Blood samples were taken 2 weeks prior to the study, at baseline and at weeks 2, 4 and 8. Blood was drawn from the antecubital vein in the morning after an overnight fast. The serum was separated immediately and was kept frozen at -70°C until analyzed.

Serum quercetin concentrations were analyzed using a validated method developed at our laboratory (Erlund et al, 1999). In this method potential conjugates of quercetin are hydrolyzed and therefore the results represent total quercetin (unconjugated quercetin, quercetin conjugated with glucuronic acid, sulfate or glycoside groups, and quercetin either bound or not bound to protein). In brief, quercetin conjugates were hydrolyzed by incubating 1 ml of serum with 110 µl of 0.78 M sodium acetate buffer (pH 4.8), 100 µl of 0.1 M ascorbic acid and 40 µl of a crude preparation from Helix pomatia containing 4000 U of -glucuronidase and 200 U of sulfatase activity (type HP-2, Sigma), for 17 h at 37°C. The sample was diluted with 2 ml of phosphate buffer (70 mM, pH 2.4) and added to a Bond Elut C18 solid-phase extraction column, preconditioned with 6 ml of methanol and 6 ml of phosphate buffer. The column was washed with 9 ml of phosphate buffer and 0.5 ml of water. Quercetin was eluted into a conical glass tube with 2 ml of methanol and dried. For removal of additional interferences, 1 ml of toluene-dichloromethane (80:20, v/v) and 200 µl of 5.3 M acetic acid-32 mM oxalic acid (80:20, v/v; pH 2.4) were added. The tubes were vortexed and centrifuged. The lower phase was used for HPLC analysis.

Chromatographic analysis was performed with a system consisting of an HP 1090 liquid chromatograph (Hewlett-Packard, Palo Alto, CA, USA), a Coulochem 5100A electrochemical detector with a model 5011 analytical cell (ESA Inc., Chelmsford, MA, USA) and an Inertsil ODS-3 analytical HPLC column (250´4.0 mm i.d., 5 µm; GL Sciences, Tokyo, Japan). The mobile phase consisted of 59% of methanol in phosphate buffer (70 mM, pH 2.4). The detector potential was set to 100 mV.

Quantitation of the quercetin peak was based on the standard additions method using serum standards containing 0, 10, 30, 90 or 150 µg/l of added quercetin. The reproducibility of the method was followed by analyzing a pooled reference sample with a concentration of 72 µg/l in triplicate in each run. Day-to-day variation (CV%) of the reference was 6.4%.

Statistical methods

Statistical significance of the difference between serum quercetin concentrations of the two dietary groups was assessed by analysis of covariance (ANCOVA) for repeated measures. The baseline values served as covariates and time (weeks 2, 4, 8) as repeated measure. Post-hoc comparisons were performed using contrast analysis. Whether the intake of quercetin differed between the groups at 8 weeks was tested by ANCOVA. The baseline values served as covariates. Post-hoc comparisons were performed by Tukey's test. Whether the baseline values of serum quercetin or quercetin intake differed between the two groups was tested by Student's t-test. The paired t-test was used to test the difference in quercetin intake between baseline and 8 weeks within the two groups. A P-value of less than 0.05 was considered statistically significant.

Results

Intake of quercetin

In the berry group the mean calculated intake of quercetin was significantly higher at the end of the 8 week study compared with baseline (P=0.001, paired t-test; Table 1). In the control group the intake did not change (P>0.1, paired t-test). Quercetin intake was slightly higher at baseline in the berry group compared to the control group but the difference was non-significant (P>0.1, Student's t-test). At the end of the study the intake of quercetin was significantly higher in the berry group compared with the control group (P=0.001, ANCOVA). The intake of quercetin from the background diet (when intake from berries was disregarded), did not change within groups during the study (P>0.1 ANCOVA). In the berry group the mean estimated intake of quercetin from the berries was 6.2 mg/day.

Compliance

Compliance appeared to be good in this study. No deviations from the instructions regarding diet or berry consumption were reported in the 3 day dietary records or at other times. Also, serum vitamin C concentrations increased in the berry group from 9.4±2.9 mg/l (mean±s.d.) at baseline to 11.9±2.5 mg/l at 8 weeks (P<0.001, analysis of variance for repeated measures), while no significant changes occurred in the control group. This further supports the interpretations that the subjects actually ate the berries.

Serum quercetin concentrations

Serum quercetin concentrations were significantly higher in the berry group during the berry consumption period than in the control group (P=0.039 ANCOVA with repeated measures). According to contrast analysis the differences were significant at 4 weeks (P=0.034) and 8 weeks (P=0.046). In the berry group the mean concentrations ranged between 21.4 and 25.3 µg/l between weeks 2 and 8. These values were 32-51% higher compared with the control group at the corresponding timepoints. Two weeks prior to the study and at baseline the mean concentrations of the two groups were very similar (Figure 1). At these two time-points, when the men still followed their habitual diets, the mean quercetin concentration for all subjects was 16.3±12.9 µg/l (mean±s.d.).


Discussion

The aims of this study were to investigate plasma quercetin concentrations after long-term consumption of berries and in subjects consuming their habitual diets. The effects of berry consumption and antioxidant supplementation on serum antioxidant capacity were also studied, but the results have been published elsewhere (Marniemi et al, 2000).

Daily consumption of 100 g of berries (black currants, lingonberries and bilberries) significantly increased fasting serum quercetin concentrations. Compared with the control group, the concentrations were 32-51% higher in the berry group during the berry consumption period. The increase in serum quercetin was similar to or higher than what was previously reported in 12 men consuming 375 ml of black tea or 750 ml of red wine for 4 days, but less than half of what was found when the subjects consumed 50 g of fried onions (de Vries et al, 2001). However, in our study the serum samples were taken after an overnight fast, while in the study by de Vries et al they were taken twice during the last day they consumed the quercetin-containing foods. Therefore, the results are not quite comparable, but they do suggest that more quercetin reaches the systemic circulation after consumption of 100 g of these berries than after consumption of 750 ml of red wine or 375 ml of black tea.

Few reports have been published regarding plasma quercetin concentrations in subjects following their habitual diets. In most studies, the subjects have followed a flavonoid-restricted diet prior to ingestion of quercetin-containing foods or supplements. In this study, the prestudy concentrations of quercetin in all subjects were 16±13 µg/l (mean±s.d.), which is similar or slightly lower than what we found previously in subjects consuming their habitual diets; in 100 healthy university students and employees the concentration was 24±17 µg/l (Freese et al, 2002) and in 37 healthy female hospital employees it was 16±24 µg/l (Erlund et al, 2002). The results are similar to those of Noroozzi et al (2000), who reported plasma concentrations of 23±4 µg/l (mean±s.e.m.) in five men and five women with diabetes, also following their habitual diets.

The duration of supplementation was 8 weeks. This time was considered long enough to allow changes to occur in antioxidant capacity. A shorter time-period would have sufficed to reach a steady-state concentration for quercetin. Usually, the time needed for this is four to five elimination half-lives. For quercetin, half-lives between 15 and 28 h have been reported (Hollman et al, 1997; Erlund et al, 1999), which indicates that steady-state levels are reached within 3-6 days. However, to our knowledge, no studies actually showing that this applies to quercetin have been performed and most bioavailability or pharmacokinetics studies with quercetin have involved one-time ingestion of quercetin-rich foods or supplements. For some compounds following nonlinear kinetics, the kinetic behavior changes during long-term administration or is disproportional to what is expected based on single-dose studies (Ludden, 1991). Therefore, a longer study time is an advantage when investigating a compound with poorly known kinetic behavior. In this study, serum quercetin concentrations remained relative stable during the berry consumption period.

Based on the results of this study no conclusions can be made about which of the berries contributed most to the increase in serum quercetin concentrations. Preliminary studies in our laboratory have shown that quercetin is bioavailable from all of the berries used in this study (data not shown), but whether the bioavailability of the compound is different from the different berries is not known. Lingonberries, black currants and bilberries contain partly different quercetin glycosides (Kühnau, 1976; Koeppen & Herrmann, 1977, Häkkinen & Auriola, 1998) and no information is available on the bioavailability of for instance quercetin arabinosides. Also, differences in the distribution of quercetin in the different compartments of berries and the thickness of the skin could affect its availability from berries.

The estimated intake of quercetin from the berries was 6.2 mg/day. Intake was calculated from a food database, to which values for quercetin concentrations had been added. The calculated intake values are rough estimations, because the quercetin concentrations of, for instance, black currants vary a great deal depending on the cultivar, ripeness and growing conditions (Mikkonen et al, 2001). Furthermore, the berries used in this study were purchased from a berry dealer (Pakkasmarja Ltd), after which they were stored at -20°C for 7-8 months until they were consumed. Reductions of 18, 25 and 19% have been reported to occur for black currant, bilberry and lingonberry during storage at -20°C for 6 months (Häkkinen, 2000) and it is likely that similar degradation of quercetin occurred during storage in this study. Therefore, the plasma quercetin concentrations after berry consumption would probably have been higher if fresh berries had been used. However, the harvesting season for each of the berries is only a few weeks in the autumn and most berries are eaten from the freezer. Our approach was therefore a more realistic one.

In general, berries are an important source of quercetin in the Finnish diet. According to a recent estimate, which was based on Finnish 1998 annual food consumption data, berries account for 25% of the total quercetin intake (9.5 mg/day; Häkkinen, 2000). In the Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Study 8.3% of the total quercetin intake (7.4 mg) in Finnish middle-aged male smokers was estimated to originate from berries (Hirvonen, 2001). In other Scandinavian countries, berries probably contribute similarly or less to the total quercetin intake. The annual per capita consumption of fresh berries in Norway, Finland, Denmark, Iceland and Sweden has been estimated as 9.6, 9.6, 3.4, 2.2 and 1.3 kg, respectively (Johansson et al, 1998). However, at least in Finland, consumption varies between persons living in different parts of the country (Kleemola et al, 1994) and between urban and rural areas. In the 1992 Dietary Survey of Finnish Adults, the median daily consumption of unprocessed berries was 14 and 26 g for men and women, respectively (M-L Ovaskainen, personal communication). In the 90% quartile, the corresponding values were 93 and 111 g, which are similar to the amount consumed in this study.

Increased intake of berries can be recommended because, in addition to quercetin, they are rich sources of many other potentially beneficial compounds as well, and are low in fat and energy. Anthocyanins, for instance, are present in berries in high concentrations and are potent antioxidants in vitro. However, their bioavailability appears to be quite low and they are excreted very rapidly (Murkovic et al, 2000). Therefore compounds such as quercetin and phenolic acids, together with vitamin C, could play a more important role in the possible health effects of berries.

In conclusion, the results of this study indicate that berries are a good source of bioavailable quercetin, and that in the Finnish population the mean fasting serum quercetin concentration is about 20 µg/l.


Acknowledgements

We would like to thank Dr M-L Ovaskainen for providing data on quercetin concentrations of foods and for calculating berry consumption in the 1992 Dietary Survey of Finnish Adults.


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Figures

Figure 1 Serum quercetin concentrations (mean±s.e.m.) in middle-aged men consuming 100 g/day of berries as a part of their habitual diets (n=20) or their habitual diets (n=20). * P<0.05 between groups at the time-point, contrast analysis.


Tables

Table 1 The intake of quercetin (mg/day, mean±s.e.m.) assessed by 3 day food records

Received 9 October 2001; revised 17 April 2002; accepted 18 April 2002

January 2003, Volume 57, Number 1, Pages 37-42

quercetin-C, kanker, voedingstoffen, rode bessen, ellagic-acid


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Ellagic-acid, een natuurlijke >> Ellagic-acid (Quercetin-C), >> Ellagic acid, een natuurlijk >>